Rotaviruses are the primary cause of severe dehydrating diarrhea in infants and young children. The mortality due to rotavirus infection is significant, globally causing approximately 500,000 deaths each year. Due to its importance as a human pathogen, an important overall goal of the Laboratory remains the development of new vaccines, the improvement of existing vaccines, and the identification of other methods for preventing and treating rotavirus disease. Accomplishing this goal would be helped by a more complete understanding of the molecular biology of the virus. In particular, by defining events in the replication and packaging of the segmented double-stranded RNA genome of these viruses, we should gain the information necessary to attenuate pathogenic strains of rotavirus and thereby to molecularly engineer new candidate vaccines. The primary focus of this project is to characterize fundamental events in the replication of the rotavirus genome which then can be used to develop a reverse genetics system for manipulating the genetic information of the virus. Specifically, this project seeks to identify cis-acting signals that are important for the packaging, replication and expression of rotavirus messenger (m)RNAs. The project also seeks to characterize the structure and function of those viral proteins involved in these processes. These aims will be accomplished by a combination of procedures, which include (i) analysis of the replication and translation efficiencies of mutated viral mRNAs in cell-free systems, (ii) computer modeling and structural analysis (e.g., RNAse mapping, NMR spectroscopy) of the recognition signals in the mRNAs, (iii) characterization of the enzymatic and structural properties (e.g., analytical ultracentrifugation, X-ray crystallography, cryo-electron microscopy, CD spectroscopy) of recombinant viral proteins, and (iv) elucidation of the specificity and targets of the viral RNA-binding proteins by gel mobility shift assay and RNA-protein cross-linking. Studies performed in the last year have provided insight into (i) the function of the rotavirus packaging protein NSP2 in the synthesis of the viral genome, (ii) the recognition and replication of viral RNA by the viral RNA-dependent RNA polymerase, and (iii) the location and properties of translation enhancers in rotavirus RNAs that promote protein expression.